The isolation of differentially expressed cDNA clones from the filarial nematode <i>Brugia pahangi</i>

A cDNA library constructed from 3 day post-infective L3 of the filarial nematode Brugia pahangi was screened by differential hybridization with cDNA probes prepared from different life-cycle stages. Five cDNA clones hybridizing selectively to the mosquito-derived L3 probe were isolated and characterized. Northern blot analysis of 4 of the clones confirmed that each was most highly expressed in the mosquito-derived L3. The expression of each mRNA during parasite development in the mosquito vector was investigated using RT-PCR, and all were shown to be abundant in the immature L3. Four of the 5 cDNAs cloned coded for structural proteins: 2 cuticular collagens, and the muscle proteins tropomyosin and troponin. Further studies on troponin using an antiserum raised to the recombinant protein demonstrated that the protein, unlike the mRNA, was present in all life-cycle stages examined, while immunogold labelling demonstrated that it was localized to the muscle blocks

The NF-κB subunit c-Rel regulates Bach2 tumour suppressor expression in B-cell lymphoma

The REL gene, encoding the NF-κB subunit c-Rel, is frequently amplified in B-cell lymphoma and functions as a tumour-promoting transcription factor. Here we report the surprising result that c-rel–/– mice display significantly earlier lymphomagenesis in the c-Myc driven, Eμ-Myc model of B-cell lymphoma. c-Rel loss also led to earlier onset of disease in a separate TCL1-Tg-driven lymphoma model. Tumour reimplantation experiments indicated that this is an effect intrinsic to the Eμ-Myc lymphoma cells but, counterintuitively, c-rel–/– Eμ-Myc lymphoma cells were more sensitive to apoptotic stimuli. To learn more about why loss of c-Rel led to earlier onset of disease, microarray gene expression analysis was performed on B cells from 4-week-old, wild-type and c-rel–/– Eμ-Myc mice. Extensive changes in gene expression were not seen at this age, but among those transcripts significantly downregulated by the loss of c-Rel was the B-cell tumour suppressor BTB and CNC homology 2 (Bach2). Quantitative PCR and western blot analysis confirmed loss of Bach2 in c-Rel mutant Eμ-Myc tumours at both 4 weeks and the terminal stages of disease. Moreover, Bach2 expression was also downregulated in c-rel–/– TCL1-Tg mice and RelA Thr505Ala mutant Eμ-Myc mice. Analysis of wild-type Eμ-Myc mice demonstrated that the population expressing low levels of Bach2 exhibited the earlier onset of lymphoma seen in c-rel–/– mice. Confirming the relevance of these findings to human disease, analysis of chromatin immunoprecipitation sequencing data revealed that Bach2 is a c-Rel and NF-κB target gene in transformed human B cells, whereas treatment of Burkitt's lymphoma cells with inhibitors of the NF-κB/IκB kinase pathway or deletion of c-Rel or RelA resulted in loss of Bach2 expression. These data reveal a surprising tumour suppressor role for c-Rel in lymphoma development explained by regulation of Bach2 expression, underlining the context-dependent complexity of NF-κB signalling in cancer

Sterically stabilized diblock copolymer nanoparticles enable efficient preparation of non-aqueous Pickering nanoemulsions

We report the first example of a non-aqueous Pickering nanoemulsion, which comprises glycerol droplets dispersed in mineral oil. The droplet phase is stabilized by hydrophobic sterically stabilized poly(lauryl methacrylate)-poly(benzyl methacrylate) nanoparticles which are prepared directly in mineral oil using polymerization-induced self-assembly. First, a glycerol-in-mineral oil Pickering macroemulsion with a mean droplet diameter of 2.1 ± 0.9 μm is prepared via high-shear homogenization using excess nanoparticles as an emulsifier. Then, this precursor macroemulsion is subjected to high-pressure microfluidization (a single pass at an applied pressure of 20,000 psi) to produce glycerol droplets of approximately 200-250 nm diameter. Transmission electron microscopy studies indicate preservation of the distinctive superstructure produced by nanoparticle adsorption at the glycerol/mineral oil interface, thus confirming the Pickering nature of the nanoemulsion. Glycerol is sparingly soluble in mineral oil, thus such nanoemulsions are rather susceptible to destabilization via Ostwald ripening. Indeed, substantial droplet growth occurs within 24 h at 20 °C, as judged by dynamic light scattering. However, this problem can be suppressed by dissolving a non-volatile solute (sodium iodide) in glycerol prior to formation of the nanoemulsion. This reduces diffusional loss of glycerol molecules from the droplets, with analytical centrifugation studies indicating much better long-term stability for such Pickering nanoemulsions (up to 21 weeks). Finally, the addition of just 5% water to the glycerol phase prior to emulsification enables the refractive index of the droplet phase to be matched to that of the continuous phase, leading to relatively transparent nanoemulsions

Shape-shifting thermoresponsive block copolymer nano-objects

In this Feature Article, we review our recent progress in the design of shape-shifting thermoresponsive diblock copolymer nano-objects, which are prepared using various hydroxyl-functional (meth)acrylic monomers (e.g. 2‑hydroxypropyl methacrylate, 4‑hydroxybutyl acrylate or hydroxybutyl methacrylate) to generate the thermoresponsive block. Unlike traditional thermoresponsive polymers such as poly(N-isopropylacrylamide), there is no transition between soluble and insoluble polymer chains in aqueous solution. Instead, thermally driven transitions between a series of copolymer morphologies (e.g. spheres, worms, vesicles or lamellae) occur on adjusting the aqueous solution temperature owing to a subtle change in the partial degree of hydration of the permanently insoluble thermoresponsive block. Such remarkable self-assembly behavior is unprecedented in colloid science: no other amphiphilic diblock copolymer or surfactant system undergoes such behavior at a fixed chemical composition and concentration. Such shape-shifting nano-objects are characterized by transmission electron microscopy, dynamic light scattering, small-angle X-ray scattering, rheology and variable temperature 1H NMR spectroscopy. Potential applications for this fascinating new class of amphiphiles are briefly considered

Imaging of 3D tissue-engineered models of oral cancer using 890 and 1300 nm optical coherence tomography

© 2015, Nizhny Novgorod State Medical Academy. All rights reserved. Optical coherence tomography (OCT) generates its primary form of contrast from elastic backscatter. It is now the gold standard technique for retinal screening and is emerging rapidly in cardiovascular research however it remains a research goal to establish it to the same degree in epithelial cancer detection and diagnosis. In this report we compare two different OCT systems: an 890 nm spectrometer-based OCT system with 2.5 µm axial resolution and a 1300 nm swept-source OCT system with 7.5 µm axial resolution to determine the effect of these different OCT parameters on the endogenous backscatter contrast of dysplastic/malignant oral mucosa models relative to normal mucosa models. Tissueengineered oral mucosa models constructed with a dysplastic cell line (DOK), a malignant cell line (Cal27) and normal cell were imaged with both of these OCT platforms and comparisons made with regard to apparent epithelial thickness and the visibility of the epithelium relative to the underlying stroma. For the Cal27’s, hematoxylin and eosin staining confirmed the formation of a keratinized layer superficial to a thickened layer of viable cells on top of the stroma. The keratinized layer presented as a hyperreflective thickened layer superficial to a darker region on both OCT platforms. The keratinized layer caused a steep fall in signal at 890 nm, making it difficult to visualise underlying structures, whereas 1300 nm OCT clearly visualized both the epithelial cells and the stroma lying beneath. For the DOK cells, hematoxylin and eosin staining confirmed the formation of an epithelial layer frequently presenting an abnormal morphology especially at the epidermal/stromal junction, with features such as infiltrating, bulbous rete pegs. These were more clearly visualized under 890 nm OCT. These observations show that 890 nm OCT retains some of its known advantages of higher contrast between anatomical tissue layers when used to observe dysplastic and malignant 3D oral mucosa constructs. However 1300 nm OCT is confirmed to possess a greater ability to image the full thickness of the model epithelia and in particular it is more suited to imaging through the keratinized layer

Synthesis of poly(stearyl methacrylate)-poly(2-hydroxypropyl methacrylate) diblock copolymer nanoparticles via RAFT dispersion polymerization of 2-hydroxypropyl methacrylate in mineral oil

Poly(stearyl methacrylate)-poly(2-hydroxypropyl methacrylate) (PSMA-PHPMA) diblock copolymer nanoparticles are synthesized via reversible addition–fragmentation chain transfer (RAFT) dispersion polymerization of 2-hydroxypropyl methacrylate (HPMA) in mineral oil at 90 °C. The relatively short PSMA precursor (mean degree of polymerization = 9) remains soluble in mineral oil, whereas the growing PHPMA block quickly becomes insoluble, resulting in polymerization-induced self-assembly (PISA). Relatively high HPMA monomer conversions (≥98%) were achieved within 70 min as confirmed by in situ1H NMR spectroscopy studies, while gel permeation chromatography (GPC) analyses indicated high blocking efficiencies and relatively narrow molecular weight distributions (Mw/Mn ≤ 1.37) for all PISA syntheses. Depending on the precise synthesis conditions, this PISA formulation can produce diblock copolymer spheres, worms or vesicles; a pseudo-phase diagram has been constructed to enable reproducible targeting of each pure phase. Thus this is a rare example of the use of a commercially available polar monomer for PISA syntheses in non-polar media that offers access to the full range of copolymer morphologies. The resulting nanoparticles were characterized using dynamic light scattering (DLS), transmission electron microscopy (TEM), oscillatory rheology and small-angle X-ray scattering (SAXS). Interestingly, PSMA9-PHPMA70 worms undergo an unusual (partial) worm-to-vesicle transition at elevated temperature. Finally, PSMA9-PHPMA50 spheres were evaluated as putative Pickering emulsifiers. Using lower water volume fractions produced water-in-oil (w/o) emulsions after high shear homogenization, as expected. However, using higher water volume fractions, shear rates or copolymer concentrations favored the formation of w/o/w Pickering double emulsions

How do charged end-groups on the steric stabilizer block influence the formation and long-term stability of Pickering nanoemulsions prepared using sterically stabilized diblock copolymer nanoparticles?

Reversible addition–fragmentation chain transfer (RAFT) solution polymerization is used to prepare well-defined poly(glycerol monomethacrylate) (PGMA) chains bearing carboxylic acid, tertiary amine, or neutral end-groups. Each of these PGMA precursors was then chain-extended in turn via RAFT aqueous emulsion polymerization of 2,2,2-trifluoroethyl methacrylate to form spherical nanoparticles as confirmed by transmission electron microscopy (TEM) analysis. Dynamic light scattering studies indicated an intensity-average diameter of approximately 25 nm. Aqueous electrophoresis measurements confirmed that the amine-functional nanoparticles became cationic at low pH owing to end-group protonation. In contrast, carboxylic acid-functional nanoparticles became appreciably anionic at pH 10 owing to end-group ionization. Finally, nanoparticles bearing neutral end-groups exhibited zeta potentials close to zero over a range of solution pH. High-shear homogenization of n-dodecane in the presence of such sterically stabilized nanoparticles led to the formation of oil-in-water Pickering macroemulsions with volume-average diameters of 20–30 μm. High-pressure microfluidization was then used to prepare the three corresponding Pickering nanoemulsions. Each Pickering nanoemulsion was characterized by analytical centrifugation and TEM studies of the dried nanoemulsion droplets confirmed their original nanoparticle superstructure. The nanoparticle adsorption efficiency at the oil–water interface was assessed by gel permeation chromatography (using a UV detector) for each nanoparticle type at both pH 3 and 7. Nanoparticles with charged end-groups exhibited relatively low adsorption efficiency, whereas up to 90% of the neutral nanoparticles were adsorbed onto the oil droplets. This observation was supported by small-angle X-ray scattering experiments, which indicated that the packing efficiency of neutral nanoparticles around oil droplets was higher than that of nanoparticles bearing charged end-groups. Analytical centrifugation was used to evaluate the colloidal stability of the aged Pickering nanoemulsions. Pickering nanoemulsions stabilized with nanoparticles bearing charged end-groups proved to be significantly less stable than those prepared using neutral end-groups

Synthesis of diblock copolymer spheres, worms and vesicles via RAFT aqueous emulsion polymerization of hydroxybutyl methacrylate

RAFT aqueous emulsion polymerization of hydroxybutyl methacrylate using a poly(glycerol monomethacrylate) precursor leads to diblock copolymer spheres, worms or vesicles. A pseudo-phase diagram is constructed and the vesicles are briefly evaluated as a Pickering emulsifier

The extraordinary outburst in the massive protostellar system NGC 6334 I-MM1 : strong increase in mid-infrared continuum emission

Financial support for this work was provided by NASA through award #07_0156 issued by USRA. Based in part on observations collected at the European Organisation for Astronomical Research in the Southern Hemisphere under ESO program 089.C-0852(A).In recent years, dramatic outbursts have been identified toward massive protostars via infrared and millimeter dust continuum and molecular maser emission. The longest lived outburst (>6 yr) persists in NGC 6334 I-MM1, a deeply embedded object with no near-IR counterpart. Using FORCAST and HAWC+ on SOFIA, we have obtained the first mid-IR images of this field since the outburst began. Despite being undetected in pre-outburst ground-based 18 μm images, MM1 is now the brightest region at all three wavelengths (25, 37, and 53 μm), exceeding the UCHII region MM3 (NGC 6334 F). Combining the SOFIA data with ALMA imaging at four wavelengths, we construct a spectral energy distribution of the combination of MM1 and the nearby hot core MM2. The best-fit Robitaille radiative transfer model yields a luminosity of (4.9 ± 0.8) × 104 L⊙. Accounting for an estimated pre-outburst luminosity ratio MM1:MM2 = 2.1 ± 0.4, the luminosity of MM1 has increased by a factor of 16.3 ± 4.4. The pre-outburst luminosity implies a protostar of mass 6.7 M⊙, which can produce the ionizing photon rate required to power the pre-outburst HCHII region surrounding the likely outbursting protostar MM1B. The total energy and duration of the outburst exceed the S255IR-NIRS3 outburst by a factor of 3, suggesting a different scale of event involving expansion of the protostellar photosphere (to 20 R⊙), thereby supporting a higher accretion rate (0.0023 M⊙ yr−1) and reducing the ionizing photon rate. In the grid of hydrodynamic models of Meyer et al., the combination of outburst luminosity and magnitude (3) places the NGC 6334 I-MM1 event in the region of moderate total accretion (~0.1–0.3 M⊙) and hence long duration (~40–130 yr).PostprintPeer reviewe

Trial to encourage adoption and maintenance of a Mediterranean diet (TEAM-MED): Protocol for a randomised feasibility trial of a peer support intervention for dietary behaviour change in adults at high cardiovascular disease risk

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. Adoption of a Mediterranean diet (MD) reduces cardiovascular disease (CVD) risk. However, interventions to achieve dietary behaviour change are typically resource intensive. Peer support offers a potentially low-cost approach to encourage dietary change. The primary objective of this randomised controlled trial is to explore the feasibility of peer support versus a previously tested dietetic-led intervention to encourageMDbehaviour change, and to test recruitment strategies, retention and attrition in order to inform the design of a definitive trial. A total of 75 overweight adults at high CVD risk who do not follow a MD (Mediterranean Diet Score (MDS ≤3)) will be randomly assigned to either: a minimal intervention (written materials), a proven intervention (dietetic support, written materials and key MD foods), or a peer support intervention (group-based community programme delivered by lay peers) for 12 months. The primary end-point is change in MDS from baseline to 6 months (adoption of MD). Secondary end-points include: change in MDS from 6 to 12 months (maintenance of MD), effects on nutritional biomarkers and CVD risk factors, fidelity of implementation, acceptability and feasibility of the peer support intervention. This study will generate important data regarding the feasibility of peer support for ease of adoption of MD in an ‘at risk’ Northern European population. Data will be used to direct a larger scale trial, where the clinical efficacy and cost-effectiveness of peer support will be tested